A system for efficient assembly of HCV structural proteins into HCV-like particles (HCV-LPs) in insect cells has been developed in our laboratory. These noninfectious HCV-like particles have similar morphologic, serologic and biophysical properties as the putative virions isolated from HCV infected humans. In contrast to recombinant subunit vaccines, the viral proteins of HCV-like particles may be presented in a native, virion-like conformation and may therefore be superior in eliciting a protective humoral and cellular immune response. The humoral and cellular immunogenicity of the HCV-LP had previously been demonstrated in the mouse and baboon models. In addition, we demonstrated the immunogenicity and induction of protective immunity by HCV-LP in chimpanzees. Our study suggests that HCV-LP immunization induces strong HCV-specific cellular immune responses and confers partial protection against HCV challenge in the chimpanzee model. The purity of HCV-LP after our current purification procedure is only about 10%, which needs to be significantly improved if this approach is going to be developed clinically as a vaccine candidate. Based on what we know about the structural features of HCV envelope proteins in the viral particles, we engineered a histidineX6 tag at the N-terminus of E2 protein, which allows surface exposure of this tag. The histidine sequences should not interfere with the structural assembly of HCV and would facilitate affinity purification. Expression of this tagged E2 in the context of HCV-LP demonstrated feasibility of this approach and further work is being performed to improve HCV-LP purification. In an effort to improve and broaden the immunogenicity of HCV-LP, we report the generation of novel chimeric hepatitis C virus-like particles carrying HCV nonstructural protein sequences with T-cell epitopes important for induction of protective immunity. Six highly-conserved HCV CD8+ T-cell nonstructural epitopes associated with viral clearance in humans were synthesized as a polytope construct and fused to the C-terminus of the E2 protein of HCV-LP. Hydrophobicity of the signal peptide sequence in the C-terminus of E2 was reduced and the polytope sequence was confirmed. Chimeric HCV-LP carrying the HCV nonstructural polytope were amplified in insect cells, purified, and characterized biochemically, immunologically and by electron microscopy. The immunogenicity of the chimeric HCV-LP was tested in AAD transgenic mice expressing the human HLA-A2.1 molecule. Similar to HCV-LP, chimeric HCV-LP induced HCV-specific humoral and cellular immune responses against the core and envelope. In addition, chimeric HCV-LP elicited robust T-cell responses against the nonstructural epitopes. The chimeric HCV-LP polytope strategy substantially improved and broadened the immunogenicity of HCV-LP and holds promise as a vaccine candidate against HCV infection in humans.In addition, we are combining The HCV-LP approach with other modalities of immunization, such as plasmid DNA, in a prime-boost regimen. Understanding the immunological correlates associated with protective immunity following HCV re-exposure is a prerequisite for the design of effective HCV vaccines and immunotherapeutics. In this study we performed a comprehensive analysis of the innate and adaptive immunity following HCV re-exposure of two chimpanzees that had previously recovered from HCV-JFH1 infection. One of the chimpanzees, CH10274, became protected from active viremia by repeatedly challenges with homologous JFH1cc and developed neutralizing antibodies, but was later infected with high-level viremia by a heterologous challenge with H77 virus that persisted for more than one year. The other chimpanzee, CH10273 was protected from a similar, heterologous H77 challenge without any evidence of neutralizing antibodies. Peripheral HCV-specific T cell responses were present in both chimpanzees after challenges and interestingly the overall magnitude of response was lower in uninfected CH10273, which, however, exhibited a more robust CD8+ T cell response. CH10273 showed higher hepatic expression of CD8 and CD56 (natural killer) markers than CH10274 did shortly after inoculation with H77. The heightened T cell response was associated with an enhanced hepatic production of interferons (both type I and II) and interferon-stimulated genes (ISGs) in CH10273. Therefore protection or clearance of HCV re-infection upon heterologous re-challenge depends on the activation of both intrahepatic innate and cellular immune responses. Furthermore, our results suggest that serum neutralizing antibodies may contribute to early control of viral replication and spread after homologous HCV re-challenges but fail to result in long-term protective immunity. Conclusion: Our study shows that protective immunity against HCV re-infection is orchestrated by a complex network of innate and adaptive immune responses.